An efficient selection system for packaging herpes simplex virus amplicons.
Due to their simplicity and flexibility of genomic construction, herpes simplex virus (HSV) amplicon-based vectors are attractive vehicles for gene delivery. However, a significant problem faced in the generation of amplicon stocks is the low amplicon to helper virus (A/H) ratio. In order to improve the proportion of amplicons generated, a selection system for amplicon production was developed in which the HSV thymidine kinase (TK) gene is inserted into an amplicon plasmid and an HSV mutant with both TK and glycoprotein H (gH) genes deleted is used as a helper virus. Using a protocol in which amplicon stocks are passaged 2-3 times in BHK cells of TK- and gH+ genotype in the presence of selection medium containing methotrexate, stock preparations with high A/H ratio (up to 5:1) and high amplicon titre (>1 x 10(9) infectious units/ml) were generated. In vitro characterization demonstrated that a high level of biologically functional products can be efficiently produced from these amplicon constructs.
Zhang
X
X
Cantab Pharmaceuticals Research Ltd, Cambridge, UK. xzhang@cantab.co.uk
O'Shea
H
H
Entwisle
C
C
Boursnell
M
M
Efstathiou
S
S
Inglis
S
S
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003470:Culture Media; D015870:Gene Expression; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D016296:Mutagenesis; D010957:Plasmids; D013937:Thymidine Kinase / Q000235:genetics*; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics
D003470:Culture Media; D007376:Interleukin-2; D014759:Viral Envelope Proteins; C044200:glycoprotein H, herpes simplex virus type 1; D013937:Thymidine Kinase